Method for detecting a gram-negative bacterial autoinducer molecule

ABSTRACT

The present invention relates to an immunogenic conjugate comprising a carrier molecule coupled to an autoinducer of a Gram negative bacteria. The immunogenic conjugate, when combined with a pharmaceutically acceptable carrier, forms a suitable vaccine for mammals to prevent infection by the Gram negative bacteria. The immunogenic conjugate is also used to raise and subsequently isolate antibodies or binding portions thereof which are capable of recognizing and binding to the autoinducer. The antibodies or binding portions thereof are utilized in a method of treating infections, a method of inhibiting autoinducer activity, and in diagnostic assays which detect the presence of autoinducers or autoinducer antagonists in fluid or tissue samples.

This application is a divisional of U.S. patent application Ser. No.10/121,207, filed Apr. 11, 2002, now U.S. Pat. No. 6,713,059, which is adivisional of U.S. patent application Ser. No. 09/293,687, filed Apr.16, 1999, now U.S. Pat. No. 6,395,282, which claims priority to U.S.Provisional Patent Application Ser. No. 60/082,025, filed Apr. 16, 1998,which is incorporated herein in its entirety.

GOVERNMENT SUPPORT

This research was supported by grant number AI133713-04 from theNational Institute of Health.

1. FIELD OF THE INVENTION

The present invention relates to an immunogenic conjugate comprising anautoinducer molecule of a Gram negative bacteria or a syntheticanalogize thereof linked to a carrier molecule. The present inventionalso relates to antibodies and binding portions thereof capable ofbinding the immunogenic conjugate, and vaccines for the treatment orprevention of infection by autoinducer producing bacteria

2. BACKGROUND OF THE INVENTION

Some pathogenic Gram negative bacteria produce chemical moieties knownas bacterial autoinducers (BAIs). BAIs are produced by Gram negativebacteria as a mechanism for communicating with other bacteria-when theyhave grown to a high cell density. This mechanism is known as quorumsensing.

BAIs assist in the transcriptional control of genes involved in a widerange of metabolic activities. When the bacterial population reaches acritical threshold, the concentration of BAIs also reach a concentrationsufficient to enable the BAIs to bind a group of transcription factors,known as R-proteins. Binding of the BAIs to the R-proteins triggersbinding of the newly formed BAI/R-protein complex to DNA, which theninduces transcription of a group of genes. In the gram negativepathogenic bacteria, a subgroup of the activated genes are pathogenicdeterminants.

BAIs are small, non-immunogenic, lipid-soluble molecules which arecapable of diffusing out of the bacteria, and into the environment wherethey enter host cells. BAIs share structural characteristics, inparticular, they have a homoserine lactone ring with an N-acyl sidechain. Variability between different BAIs resides primarily in thestructure of the acyl side chain.

It has been proposed that in addition to regulating transcription inbacteria, the BAIs also regulate transcription in cells of an infectedmammalian host. A list of presently known bacterial autoinducers (BAIs)and the Gram negative bacteria which produce them are identified inTable 1 below:

TABLE 1 Gram negative bacteria: Bacterial autoinducer (BAI): Aeromonashydrophila AHAI Agrobacterium tumefaciensN-(3-oxo)-octanoyl-L-homoserine lactone (OOHL) Burkholderia cepaciaN-octanoylhomoserine lactone Chromobacterium violaceumN-hexanoyl-L-homoserine lactone (HHL) Enterobacter agglomeransN-(3-oxo)-hexanoyl-L-homoserine lactone (OHHL) Erwinia stewarti OHHLErwinia carotovora OHHL Escherichia coli Structure not yet determinedNitrosomas europea OHHL Photobacterium fischeri OHHL, OOHL; OHLPseudomonas aeruginosa N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1);N-(butanoyl)-L-homoserine lactone (PAI- 2) Pseudomonas aureofaciensStructure not yet determined Rhizobium leguminosarumN-(3-hydroxy)-tetradecanoyl-L- homoserine lactone (HtDeHL) Serratialiquefaciens PAI-2 Vibrio fischeri OHHL Vibrio harveyiN-(3-hydroxy)-butanoyl-L-homoserine lactone (HBHL) Yersiniaenterocolitica OHHL, HHL

The Gram negative bacterium Pseudomonas aeruginosa is an opportunistichuman pathogen that causes infections in immunocompromised hosts. PAI-1has been shown to inhibit the proliferation of lymphocytes in vivo anddownregulates expression of tumor necrosis factor and interleukin-12(Telford et al., 1998, Infect Immun. 66(1):36-42). Pseudomonasaeruginosa frequently colonizes the lungs of individuals with cysticfibrosis (Hoiby, N., 1974, Acta Pathologica Microbiolo. Scand. Sect. B.82: 551-558; Reynolds et al., 1975, Ann. Intern. Med. 82:819-832). Thisbacterium produces a number of extracellular virulence factors includingexotoxin A, which is encoded by the toxA gene (Iglewski, B. H. andKabat, D., 1975, Proc. Natl. Acad. Sci. USA. 72:2284-2288; Iglewski etal., 1978, Proc. Natl. Acad. Sci. USA. 75:3211-3215); an elastolyticprotease encoded by the lasA gene; an elastolytic protease encoded bythe lasB gene; and an alkaline protease encoded by the aprA gene(Morihara, K. and Homma, J. Y., 1985, Bacterial Enzymes and Virulence,ed. Holder, I. A. (CRC Press, Boca Raton, Fla.) pp. 41-79; Bever, R. A.and Iglewski, B. H., 1988, J. Bacteriol. 170:4309-4313; Kessler, E. andSaffrin, M., 1988, J. Bacteriol. 170:5241-5247).

Pseudomonas aeruginosa utilizes a partially redundant quorum sensingmechanism which includes two autoinducers,N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) and.N-(butanoyl)-L-homoserine lactone (PAI-2) (see Table 1). Theseautoinducers control expression of a number of virulence factors,including the elastolytic proteases lasA and lasB, autoinducer-synthase,alkaline protease, exotoxin A and rhamnolipid synthase. (Garnbello, etal., 1993, Infection & Immunity 61:1180-84; Latifi, et al., 1996, Mol.Microbiol. 21:1137-46; Passador, et al., 1993, Science 260:1127-30;Pesci, et al., 1997, J. Bact. 179:3127-32; Seed, et. al., 1995, J. Bact.177:654-59; and Toder, et. al., 1994, Infection & Immunity 62:1320-27.)It is the production of these virulence factors which enable Pseudomonasaeruginosa to invade and induce disease in humans.

Current treatments for Gram negative bacterial infections typicallytarget surface antigens of the bacteria to make antibodies. Developmentof vaccines and diagnostic antibodies to autoinducers are hindered bythe fact that autoinducers are not only non-immunogenic, but are alsofreely diffusible through the lipid bilayer and are not covalentlyattached to the bacteria Several studies have demonstrated that anon-immunogenic bacterial capsular polysaccharide may be conjugated toan immunogenic compound to generate antibodies to the capsularpolysaccharide (Anderson, U.S. Pat. No. 4,673,574 (conjugation of afragment of a bacterial capsular polymer to a diphtheria or tetanustoxin or toxoid); Wessels et al., 1990, J. Clin. Invest. 86:1428-1433(conjugation of a polysaccharide of type III group B Streptococcus totetanus toxoid); and Schneerson et al., 1980, J. Exp. Med. 152:361-376(conjugation of H. influenzae type b capsular polysaccharide to tetanustoxoid and other carriers.)) However, in contrast to autoinducers whichare lipid diffusible, these anti-polysaccharide treatments are designedfor production of antibodies specific to surface antigens covalentlyattached to the bacteria, resulting in lysis of the bacteria.

While synthetic autoinducer analogs limit bacterial growth in vitro,this approach fails to harness the capabilities of an active immuneresponse that is a potentially long-lasting and effective therapeutic orprophylactic treatment (Pearson et al., U.S. Pat. No. 5,591,872).Furthermore, although autoinducer molecules themselves can be used indiagnostic bioassays, including bioluminescence, antibiotic production,or bacterial growth, these diagnostic assays fail to provide aprophylactic or therapeutic benefit to individuals exposed toautoinducer-producing Gram negative bacteria (Bycroft et al., U.S. Pat.No. 5,593,827).

3. SUMMARY OF THE INVENTION

The present invention relates to immunogenic conjugates comprising acarrier molecule covalently conjugated or otherwise bound to anautoinducer of a Gram negative bacteria of a compound of Formula (I):

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl, C₁-C₁₈ straight branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkyl,C₁-C₆ straight or branched alke C₁-C₆ straight or branched alkynyl, orCO₂H; and Q is CH or N; and n is 0-3 with the proviso that when n is 0,X is N—(C₁-C₆ alkyl) or N-phenyl. In a specific embodiment, the carriermolecule comprises a lysine-containing protein, preferably, includingbut not limited to bovine serum albumin, chicken egg ovalbumin, keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, and thyroglobulin.

In specific embodiments, the autoinducer is produced by a Gram negativebacteria comprising Aeromonas hydrophila, Agrobacterium tumefaciens,Burkholderia cepacia, Chromobacterium violaceum, Enterobacteragglomerans, Erwinia stewarti, Erwinia carotovora, Escherichia coli,Nitrosomas europea, Photobacterium fischeri, Pseudomonas aeruginosa,Pseudomonas aureofaciens, Rhizobium leguminosarum, Serratialiquefaciens, or Vibrio harveyi.

In specific embodiments, the autoinducer comprisesN-(3-oxododecanoyl-L-homoserine lactone, N-(butanoyl)-L-homoserinelactone, N-hexanoyl-homoserine lactone, N-(3-oxohexanoyl)-homoserinelactone, N-β(hydroxybutyryl)-homoserine lactone,N-(3-oxooctanoyl)-L-homoserine lactone, orN-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone, preferably,N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) orN-(butanoyl)-L-homoserine lactone (PAI-2).

In a specific embodiment, the carrier molecule of the immunogenicconjugate has at least one amine group, the autoinducer has an N-acylhomoserine lactone structure, and the conjugate is the reductiveamination product of the carrier molecule and the autoinducer.

The invention also relates to isolated antibodies or fragments thereofwhich specifically bind an autoinducer produced by a Gram negativebacteria In an embodiment, the autoinducer is a compound of Formula (I)(described above). In another embodiment, the autoinducer comprisesN-(3-oxododecanoyl)-L-homoserine lactone, N-(butanoyl)-L-homoserinelactone, N-hexanoyl-homoserine lactone, N-(3-oxohexanoyl)-homoserinelactone, N-β(hydroxybutyryl)-homoserine lactone,N-(3-oxooctanoyl)-L-homoserine lactone, orN-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone. In a specificembodiment, the autoinducer is N-(3-oxododecanoyl)-L-homoserine lactoneor N-(butanoyl)-L-homoserine lactone.

The invention also relates to isolated antibodies or fragments thereofwhich specifically bind an autoinducer produced by a Gram negativebacteria in which the autoinducer is covalently conjugated or otherwisebound to a carrier molecule. The carrier molecule includes but is notlimited to bovine serum albumin, chicken egg ovalbumin, keyhole limpethemocyanin, tetanus toxoid, diphtheria toxoid, and thyroglobulin.

In specific embodiments, the autoinducer which is specifically bound bythe antibodies or fragments thereof of the invention is produced by aGram negative bacteria comprising Aeromonas hydrophila, Agrobacteriumtumefaciens, Burkholderia cepacia, Chromobacterium violaceum,Enterobacter agglomerans, Erwinia stewarti, Erwinia carotovora,Escherichia coli, Nitrosomas europea, Photobacterium fischeri,Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.

The invention also relates to methods for detecting a Gram negativebacteria autoinducer in a sample comprising adding to the sample anantibody in which the antibody specifically binds the autoinducer of aGram negative bacteria of a compound of Formula (I) (described above).In an embodiment, the autoinducer is produced by a Gram negativebacteria including but not limited to Aeromonas hydrophila,Agrobacterium tuinetaciens, Burkholderia cepacia, Chromobacteriumviolaceum, Enterobacter agglomerans, Erwinia stewarti, Erwiniacarotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.

The invention also relates to methods of treating or preventing aninfectious disease in a subject comprising administering an amount of animmunogenic conjugate in which the immnuogenic conjugate comprises acarrier molecule covalently conjugated or otherwise bound to anautoinducer of a Gram negative bacteria of a compound of Formula (I)(described above); preferably; the subject is a human.

The invention also relates to methods of treating or preventing aninfectious disease in a subject comprising administering an amount of anantibody or fragment thereof which specifically binds an autoinducer ofa Gram negative bacteria of a compound of Formula (I) (described above);preferably, the subject is a human.

The invention also relates to diagnostic kits and pharmaceuticalcompositions comprising the immunogenic conjugates or antibodies orfragments thereof of the invention.

The present invention also relates to methods of inhibiting autoinduceractivity. The methods comprise contacting an effective amount of theantibody or binding portion thereof with an autoinducer under conditionseffective to bind the autoinducer in which the amount is effective totreat or prevent infection by Gram negative bacteria.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of the role of autoinducers PAI-1 and PAI-2 in thevirulence of Pseudomonas aeruginosa. Neonatal mice were infected withPseudomonas aeruginosa strains PAO1 (wild type), PAO-JP2 (bearing thelasI/rhII double deletion), PAO-JP1 (bearing lasI single deletion), andPAO-JP2/pJPP42 (bearing the lasI/rhII double deletion with plasmidexpressing lasI/rhII). The mice were sacrificed and examined forpneumonia (black bars), bacteremia (light gray bars), and mortality(dark gray bars).

FIG. 2. The effect of anti-PAI-1 murine polyclonal antibodies on an E.coli transcriptional bioassay. A 1:10 dilution of mouse serum waspreincubated with 100 pM PAI-1 prior to addition to the bioassay. “Mal1” is compound-D conjugate immune serum, “PAI-1” is PAI-1 conjugateimmune serum, “Pre” is serum from mice prior to any immunization and“control” is PAI-1 without addition of serum. Addition of immune serumexhibited a 70% inhibition in the production of β-galactosidase.

FIG. 3. The effect of anti-PAI-1 monoclonal antibodies in a Pseudomonasaeruginosa transcriptional bioassay. When an anti-PAI-1 monoclonalantibody (618.4) was added to 40 nM PAI-1 prior to addition to thebioassay an 80% inhibition in β-galactosidase production was exhibited.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for the production of immunogenicconjugates of autoinducers and immunogenic conjugates for use asvaccines or for production of antibodies useful for immunotherapy ordiagnostic assays.

Solely for ease of explanation, the description of the invention isdivided into the following sections: (A) isolation of homoserinelactones; (B) conjugation of autoinducers to immunogenic carriers; (C)generation of antibodies to BAI immunogenic conjugates; (D) therapeuticuses of immunogenic conjugates or antibodies thereto; (E) diagnosticassays.

5.1 Isolation and Synthesis of Homoserine Lactones

Autoinducers of Grain negative bacteria can be isolated and purified byvarious methods known in the art. Purification of homoserine lactonesthrough chemical methods can be used to isolate naturally producedautoinducers. The autoinducer N-(β-ketocaproyl) homoserine lactone hasbeen purified from a culture supernatant of Erwinia carotovora (Bycroftet al., U.S. Pat. No. 5,593,827, hereby incorporated by reference).Following bacterial culture centrifugation, the autoinducer is extractedfrom the supernatant using ethyl acetate, the resulting sample is mixedwith water and the ethyl acetate removed. Next, the sample is passedthrough a column containing a hydrophobic resin which is eluted with amethanol in water solution to remove the autoinducer. The methanol inwater solution, now containing the autoinducer, is concentrated byrotary evaporation. Thereafter, the autoinducer is purified using HPLCand additional rotary evaporation.

A similar process has been developed for purifying the naturallyoccurring autoinducer of Pseudomonas aeruginosa,N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) (Pearson et al., U.S.Pat. No. 5,591,872, which is hereby incorporated by reference). First,cells and culture fluid are separated by centrifugation and the culturefluid subsequently passed through a 0.2 μm pore-size filter. Thefiltered material is repeatedly extracted using alternatingethanol/ethyl acetate steps. The sample is subsequently dissolved inmethanol and purified using HPLC with reverse phase column. Additionalextraction and purification by HPLC yield the isolated autoinducerPAI-1.

Eberhard et al., 1981, Biochem. 20:2444-49, which is hereby incorporatedby reference, describes a process for chemically synthesizingN-3-oxohexanoyl)-homoserine lactone. U.S. Pat. No. 5,591,872 to Pearsonet al., establishes that the process of Eberhard et al. may also be usedto synthesize N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1), by usinga different starting material (i.e., ethyl 3-oxododecanoate rather thanethyl 3-oxohexanoate). Therefore, it may be appreciated by one ofordinary skill in the art that the procedure of Eberhard et al. may beused to synthesize a variety of autoinducer molecules and theirderivatives by utilizing different starting materials.

Compounds of the Formula (I) where L is CO₂H or CO₂-alkyl, m is 0, Y isCH₂, n is 1, Q is CH, and X is 0 may be prepared according to Scheme I.Reaction of L-homoserine lactone hydrochloride (B) with malonic acidmono tert-butylester (A) in methylene chloride at room temperature inthe presence of an organic base, such as triethylamine, and a couplingagent, such as benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate yielded the corresponding amide (C). Amide (C) wasdeprotected under acidic conditions using trifluoroacetic acid (TFA) inmethylene chloride at room temperature to yield the carboxylic acid (D).Homoserine derivatives of the formula (D) can be conjugated to amine oralcohol functionalities on the appropriate carrier protein by reactionwith the carboxylic acid moiety under standard conditions to one skilledin the art to form an amide or ester conjugate, respectively, with thecarrier protein.

Compounds of the Formula (I) where Y and L are alkyl, Q is CH, n is 1and m is 0 were prepared in a single step as outlined in Scheme 2. Thus,reaction of the sodium or lithium salts of the fatty acids 1-3 withoptically pure L-homoserine lactone hydrochloride Sigma Chemical Co.,St. Louis, Mo.) in the presence of the commercially available watersoluble coupling agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (Aldrich Chemical Company, Milwaukee, Wis.), provided thewaxy amides 5-7 in good yield. In each case, the crude products wereconveniently purified by recrystallization from ethyl acetate/hexane.

The natural form of Pseudomonas autoinducer (PA) 13 was synthesized in anovel six step sequence as outlined in Scheme 3. Standard acylation ofMeldrums acid with decanoyl chloride in the presence of pyridineprovided the acyl Meldrums species 8. Reaction of 8 with methanol underreflux conditions then provided the beta-ketoester 9 in good yield. TheC3 carbonyl group of 9 was then protected as its ketal under standardconditions and hydrolysis of the methyl ester functionality under basicconditions then afforded the carboxylate 10. Coupling of 10 andL-homoserine lactone hydrochloride under aqueous conditions in thepresence of water soluble carbodiimide 4 then provided the amide 11 ingood yield. Final deprotection of 11 was accomplished with dilute acidaffording the target keto-amide 13 (PAI). An analogous sequence wascarried out in order to prepare the keto-amide 14 bearing a thiolactonemoiety. In this sequence, optically pure L-homocysteine thiolactonehydrochloride (available from Sigma) was substituted for L-homoserinelactone hydrochloride in the carbodiimide mediated coupling step. Theresulting dioxolane-amide 12 was then deprotected with dilute acid toprovide the keto-amide 14.

Compounds of the Formula (I) where Z is CHOH, Y is CH₂, L is alkyl, n is1, Q is CH and X is 0 were prepared by reduction of the C3 keto group of13 as outlined in Scheme 4. Thus, low temperature reduction of 13 withsodium borohydride in methanol provided a diastereomeric mixture ofalcohols 15.

Compounds of the Formula (I) where X is 0, Q is N, m is 0, z is 0, andR² is straight or branched alkyl were prepared by direct acylation of 16with decanoyl chloride (Scheme 4). Deprotonation of commerciallyavailable optically pure 16 with BuLi followed by quenching theresulting anion with decanoyl chloride, provided the acylatedoxazolidinone 17 in good yield.

Compounds of the Formula (I) where X is 0, Y is CH₂, Z is C═O, Q is CH,n is 1, and L is C₁-C₁₈ straight or branched alkenyl may be prepared bythe method of Scheme 5. Careful oxidation of commercially availabletrans-4-decanal with sodium hypochlorite in the presence of sulfamicacid (as chlorine scavenger), provided crude acid 18 which was notpurified, but converted directly to the acid chloride 19 with oxalylchloride and DMF (cat.). Reaction of 19 with Meldrums acid in thepresence of pyridinie provided the acyl Meldrums species 20. Heatingcrude 20 in methanol then afforded the beta-ketoester 21 which was thenpurified by flash chromatography. Protection of the ketone carbonylgroup of 21 as its ketal followed by hydrolysis of the ester withlithium hydroxide gave the carboxylate salt 23. Coupling of 23 andL-Homoserine lactone hydrochloride in the presence of the carbodiimide 4in aqueous medium, provided the amide 24. Finally, deprotection of 24under acidic conditions gave the unsaturated keto-amide 25 in 36% yieldover 3 steps.

Compounds of the Formula (I) where X is NH, Q is CH, Y is CH₂, Z is C═O,L is alkyl, n is 1, m is 1, and Z is C═O were prepared according toScheme 6. The keto-lactam 28 was prepared as outlined in Scheme 6.Hydrolysis of 10 with lithium hydroxide followed by carbodiimidemediated coupling with aminolactam hydrochloride 26 provided theprotected lactam 27. The aminolactam hydrochloride 26 was prepared fromcommercially available (S)-(+)-2,4-Diaminobutyric acid hydrochloride(Aldrich Chemical Co.) according to the literature procedure. D. W.Adamson, J. Chem. Soc. 1943:39-41. Hydrolysis of 27 with dilute acidthen gave the target keto-lactam 28 in good overall yield.

Compounds of the Formula (I) where Y is C₁-C₆ alkenyl, m is 0, and Z isCO₂H can be made by the reaction of homoserine lactone with maleicanhydride in methylene chloride at room temperature to yield compound29. (Scheme 7). Linshitz, Y., et al., J. Amer. Chem. Soc., 77, 1265-6(1955).

The carboxylic acid moiety can then be coupled to a carrier proteincontaining a free amino or hydroxy group under standard conditions toyield the corresponding immunogenic conjugate.

Autoinducer molecules suitable for use in forming immunogenic conjugatesof the present invention are the autoinducers of Gram negative bacteriaof a compound of Formula (I) and derivatives thereof. In particular, theautoinducers used to form the immunogenic conjugate of the presentinvention are the autoinducers of the following Gram negative bacteria:Aeromonas hydrophiia, Agrobacterium tumefaciens, Burkholderia cepacia,Chromobacterium violaceum, Enterobacter agglomerans, Erwinia stewarti,Erwinia carotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liqueffaciens, and Vibrio harveyi.

Preferred autoinducer molecules are N-(3-oxododecanoyl)-L-homoserinelactone, N-(butyryl)-L-homoserine lactone, N-butanoyl-L-homoserinelactone, N-hexanoyl-homoserine lactone, N-(3-oxohexanoyl)-homoserinelactone, N-β-(hydroxybutyryl)-homoserine lactone,N-3-oxooctanoyl)-L-homoserine lactone,N-(3R-hydroxy-cis-tetradecanoyl)-L homoserine lactone, other N-acylhomoserine lactones, their derivatives and analogs.

The structures of various exemplary autoinducer molecules are shownbelow:

5.2 Bacterial Autoinducer Conjugation to a Carrier

Once the desired autoinducer is isolated, the immunogenic conjugates ofthe present invention are formed by coupling the carrier molecule to theautoinducer. Typically, these reactions are conducted by reactingavailable hydroxy or amino groups in a protein, with a compound of theformula (I) as shown below:

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched allyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl, C₁-C₁₈ straight branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkyl,C₁-C₆ straight or branched alkyl, C₁-C₆ straight or branched alkynyl, orCO₂H; and Q is CH, or N; and n is 0-3 with the proviso that when n is 0,X is N—(C₁-C₆ alkyl) or N-phenyl.

The reactions are performed under acidic or basic conditions in thepresence of a suitable coupling agent. For example, an autoinducer ofGram negative bacteria may contain a reactive ketone moiety in acompound of Formula (I). The reaction of the reactive ketone moiety of acompound of Formula (I) with a carrier molecule such as a proteincontaining a free amino group will produce a Schiff base which can thenbe reduced with sodium cyanoborohydride to produce the correspondingamine (reductive amination). Sodium cyanoborohydride is the preferredagent for the reduction of Schiff bases in the presence of other ketonemoieties present in the autoinducer compound of Formula (I).

Other reducing agents can be used to reductively aminate ketone moietiesinstead of sodium cyanoborohydride. Their suitability will depend uponthe functionalities present in the autoinducer compound of Formula (I).The alternative reducing agents include hydrogen and a catalyst ofeither sodium triacetoxyborohydride (Carson et al., 1990, TetrahedronLetters, 31, 5595), sodium borohydride (Schellenberg, 1963, J. Org.Chem., 28, 3259) alcoholic potassium hydroxide (Watanabe et al., 1974,Tetrahedron Letters, 1879) or BH₃-Pyridine (Pelter et al., 1984, J.Chem. Suc. Perkin Trans. 1:717). The reductive amination process iscarried out using the method set forth by Schwartz and Gray, “ProteinsContaining Reductively Aminated Disaccharides: Synthesis and ChemicalCharacterization,” Arch. Biochem. Biophys. 181:542-549 (1977), which ishereby incorporated by reference.

Alternatively, a carrier molecule such as a protein containing a freeamino group can be coupled to carbon-carbon multiple bonds present in acompound of Formula (I) under basic conditions to form an amineconjugate or imine conjugate in the case where addition occurs to analkyne. The free amino group of a protein could also add to anisocyanate or isothiocyanate moiety of a compound of Formula (I) toyield a urea or thiourea conjugate, respectively. Vishnyakora, et al.,Russ. Chem. Rev. 54, 1249-261 (1985). Additionally, amine conjugatecontaining a compound of Formula (I) can be made by reaction of anallylic halide moiety with a protein containing a free amino group underbasic conditions.

For example, the Pseudomonas aeruginosa autoinducer PAI-1 may beconjugated to a carrier molecule such as a lysine containing protein(i.e., bovine serum albumin). PAI-1 and the protein carrier molecule aremixed at a 1:10 molar ratio in phosphate buffer (pH 7.0). PAI-1 via theketo group, interacts with the terminal amino groups of lysines on thesurface of the protein carrier molecule to form a Schiff base. Themixture is incubated until conjugation is substantially complete. Afterconjugation, the intermediate Schiff base formed between PAI-1 and theprotein carrier molecule is reduced with sodium cyanoborohydride.Finally, the mixture is dialyzed for removal of unbound PAI-1 andresidual cyanoborohydride.

Alternatively, a BAI may be prepared by coupling a carboxylic acidmoiety of L or R² of a compound of Formula (I) with an amine of aprotein to form a peptide conjugate. As coupling reagents, one can useany standard peptide coupling activation method for carboxylic acidmoieties such as those exemplified in schemes 1-6 described earlier, orother coupling agents or methods (e.g., photocoupling) known to thoseskilled in the art.

As another alternative, the BAI may be non-covalently bound to thecarrier molecule by being absorbed to the carrier molecule usingtechniques commonly known in the art.

Suitable carrier molecules are those which are safe for administrationto mammals and immunologically effective as carriers. Safety wouldinclude the absence of primary toxicity and minimal risk of allergiccomplications. Preferred carrier molecules are bovine serum albumin,chicken egg ovalbumin, keyhole limpet hemocyanin, tetanus toxoid,diphtheria toxoid, thyroglobulin, and other lysine containing proteins.Each of the preferred carrier molecules fulfill these criteria, becausethey are non-toxic and the incidence of allergic reaction is well known.

The formation of immunogenic conjugates comprising an autoinducer of aGram negative bacteria and a carrier molecule enables a wide array oftherapeutic and/or prophylactic agents and diagnostic procedures for,respectively, treating or preventing infection by Gram negative bacteriaand detecting the presence of autoinducer molecules produced thereby.

5.3. Generation of Antibodies to BAI Immunogenic conjugates

Another aspect of the present invention relates to isolated antibodiesor binding portions thereof which specifically bind an autoinducer ofthe present invention. The present invention also encompassespharmaceutical compositions comprising the antibodies or bindingportions thereof and a pharmaceutically acceptable carrier.

An autoinducer immunogenic conjugate or autoinducer or derivativethereof, may be used as an immunogen to generate antibodies whichrecognize the said immunogen. The antibodies include but are not limitedto polyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above.

The antibodies or binding portions thereof or compositions containingthe same, are useful in treating mammals, preferably humans, exposed toor otherwise infected with a autoinducer producing Gram negativebacteria Methods of treatment using the compositions of the presentinvention include but are not limited to passive immunotherapy, idiotypevaccination, etc. Methods of treatment encompassed by the presentinvention comprise administration of a therapeutically effective amountof the antibody or binding, portion thereof in which the antibody orbinding portion thereof is capable of binding an autoinducer of a Gramnegative bacteria.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to an autoinducer immunogenic conjugate. For theproduction of antibodies, various host animals can be immunized byinjection with an immunogenic conjugate, or a synthetic version, orderivative (e.g., fragment) thereof. Such host animals include but arenot limited to rabbits, mice, rats, etc. Adjuvants may be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Various procedures for raising polyclonal antibodies aredescribed in E. Harlow, et al., editors, Antibodies: A Laboratory Manual(1988), which is hereby incorporated by reference. For an illustrativeexample, see section 6.2, infra.

For preparation of monoclonal antibodies directed toward an autoinduceror analog thereof, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique of Kohler and Milstein (1975, Nature256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Human antibodies may be used and can be obtained by using humanhybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030)or by transforming human B cells with EBV virus in vitro (Cole et al.,1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp.77-96). In addition, monoclonal antibodies can be produced in germ-freeanimals utilizing recent technology (PCT/US90/02545).

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., 1984, Proc. Natl. Acad. Sci., 81,6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al.,1985, Nature, 314, 452-454, incorporated herein by reference in theirentirety) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used, forexample, the genes from a mouse antibody molecule specific for anautoinducer can be spliced together with genes from a human antibodymolecule of appropriate biological activity. A chimeric antibody is amolecule in which different portions are derived from different animalspecies, such as those having a variable region derived from a murinemAb and a human immunoglobulin constant region. (See, e.g., Cabilly etal., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397,which are incorporated herein by reference in their entirety.)

In addition, techniques have been developed for the production ofhumanized antibodies. (See, e.g., Queen, U.S. Pat. No. 5,585,089 andWinter, U.S. Pat. No. 5,225,539, which are incorporated herein byreference in their entirety.) An immunoglobulin light or heavy chainvariable region consists of a “framework” region interrupted by threehypervariable regions, referred to as complementarity determiningregions (CDRs). The extent of the framework region and CDRs have beenprecisely defined (see, “Sequences of Proteins of ImmunologicalInterest”, Kabat, E. et al., U.S. Department of Health and HumanServices (1983), incorporated herein by reference in their entirety).Briefly, humanized antibodies are antibody molecules from non-humanspecies having one or more CDRs from the non-human species and aframework region from a human immunoglobulin molecule.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242, 423-426;Huston, et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879-5883; andWard, et al., 1989, Nature 334, 544-546, incorporated herein byreference in their entirety) can be adapted to produce single chainantibodies against an immunogenic conjugate of the present invention.Single chain antibodies are formed by lining the heavy and light chainfragments of the Fv region via an amino acid ridge, resulting in asingle chain polypeptide.

In another embodiment, the methods of the present invention encompassuse of antibody fragments comprising the idiotype of the whole antibody.Such fragments include but are not limited to: the F(ab′)₂ fragments,which can be produced by pepsin digestion of the antibody molecule andthe Fab fragments, which can be generated by reducing the disulfidebridges of the F(ab′)₂ fragments (J. Goding, Monoclonal Antibodies:Principles and Practice pp.98-118 (N.Y. Academic Press 1983), which isincorporated herein by reference). Alternatively, the Fab fragments canbe generated by treating the antibody molecule with papain and areducing agent. Alternatively, Fab expression libraries may beconstructed (Huse, et al., 1989, Science, 246:1275-1281, incorporatedherein by reference in its entirety) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

In an embodiment of the invention, molecules comprising the bindingportion of antibodies which specifically bind an autoinducer or theepitope of an autoinducer may be used in the methods of the invention.Such molecules include peptides, derivatives and analogs thereof, andpeptide mimetics.

The autoinducer specific antibodies may be isolated by standardtechniques known in the art such as immunoaffinity chromatography,centrifugation, precipitation, etc. Screening for the desired antibodycan be accomplished by techniques known in the art including but notlimited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immonodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complementfixation-assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled.

The foregoing antibodies can be used for treating or preventing diseasescaused by the auto inducer producing Gram negative bacteria or fordetecting and measuring the activity of the autoinducer of theinvention, e.g., for imaging these autoinducers, measuring levelsthereof in appropriate physiological samples, in diagnostic assays, etc.as discussed in section 5.5 infra.

The antibodies, generated by the vaccine formulations of the presentinvention can also be used in the production of antiidiotypic antibody.The anti idiotypic antibody can then in turn be used for immunization,in order to produce a subpopulation of antibodies that bind the initialantigen of the pathogenic microorganism (Jerne, 1974, Ann. Immunol.(Paris) 125c:373; Jerne, et al., 1982, EMBO J. 1:234).

5.4 Therapeutic Uses of Immunogenic Conjugates or Antibodies Thereto

Autinducer immunogenic conjugates and antibodies which specifically bindan autoinducer by a Gram negative bacteria can be used for treating orpreventing an infectious disease caused by a Gram negative bacteria.

5.4.1 Administration and Formulation

Immunogenic conjugates comprising an autoinducer of a Gram negativebacteria of a compound of Formula (I) coupled to a carrier molecule canbe used as vaccines for immunization against said autoinducer producingGram negative bacteria. The vaccines, comprising the immunogenicconjugate in a pharmaceutically acceptable carrier, are useful in amethod of immunizing mammals, preferably humans, for treatment orprevention of infections by the said autoinducer producing Gram negativebacteria.

The antibodies, generated against the autoinducer immunogenic conjugateof the present invention by immunization with the autoinducerimmunogenic conjugate can be used in passive immunotherapy andgeneration of antiidiotypic antibodies, for treating or preventinginfectious disease caused by a Grain negative bacteria.

In a specific embodiment, an immunogenic conjugate comprising thePseudomonas aeruginosa autoinducer PAI-1 (see Table 1) is administeredas a vaccine to Pseudomonas aeruginosa. In another embodiment, animmunogenic conjugate comprising the Pseudomonas aeruginosa autoinducerPAI-2 (see Table 1) is administered as a vaccine. In another embodiment,an immunogenic conjugate comprising PAI-1 is administered before,during, or after administration of an immunogenic conjugate comprisingPAI-2. In another embodiment, an autoinducer immunogenic conjugatevaccine is administered before, during or after administration of aneffective amount of an antibody or binding portion thereof which hasbeen raised against an immunogenic conjugate of the type describedabove.

In an embodiment, an antibody or binding portion thereof whichspecifically binds an immunogenic conjugate comprising PAI-1 isadministere . In another embodiment, an antibody or binding portionthereof which specifically binds PAI-2 is administered. In anotherembodiment the antibody which specifically binds PAI-1 is administeredbefore, during or after administration of an antibody which speci icallybinds PAI-2.

Methods of administration of the compositions of the invention. (i.e.,BAI immunogenic conjugate vaccine or antibodies specific to the BAIimmunogenic conjugates) include but are not limited to oral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal routes, and scarification (scratching through the top-layersof skin, e.g., using a bifurcated needle).

The patient to which the composition is administered is preferably amammal, including but not limited to cows, horses, sheep, pigs, fowl(e.g. chickens), goats, cats, dogs, hamsters, mice and rats. In apreferred embodiment the subject is a human.

The formulations of the invention comprise an effective immunizingamount of one Or more autoinducer immunogenic conjugate or antibodythereto and a pharmaceutically acceptable carrier or excipient. Subunitvaccines comprise an effective immunizing amount of one or more Antigensand a pharmaceutically acceptable carrier or excipient. Pharmaceuticallyacceptable carriers are well known in the art and include but are notlimited to saline, buffered saline, dextrose, water, glycerol, sterileisotonic aqueous buffer, and combinations thereof. One example of suchan acceptable carrier is a physiologically balanced culture mediumcontaining one or more stabilizing agents such as stabilized, hydrolyzedproteins, lactose, etc. The carrier is preferably sterile. Theformulation should suit the mode of administration.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc.

Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampule or sachette indicating the quantity of active agent. Where thecomposition is administered by injection, an ampule of sterile diluentcan be provided so that the ingredients may be mixed prior toadministration.

Peptides, derivatives and analogs thereof, and peptide mimetics thatspecifically bind an autoinducer can be produced by various methodsknown in the art, including, but not limited to solid-phase synthesis orby solution (Nakanishi et al., 1993, Gene 137:51-56; Merrifield, 1963,J., Am. Chem. Soc. 15:2149-2154; Neurath, H. et al., Eds., The Proteins,Vol II, 3d Ed., p. 105-237, Academic. Press, New York, N.Y. (1976),incorporated herein in their entirety by reference).

Suitable carriers include lubricants and inert fillers such as lactose,sucrose, or cornstarch. In another embodiment, these compounds aretableted with conventional tablet bases such as lactose, sucrose, orcornstarch in combination with binders like acacia, gum gragacanth,cornstarch, or gelatin; disintegrating agents such as cornstarch, potatostarch, or alginic acid; a lubricant like stearic acid or magnesiumstearate; and sweetening agents such as sucrose, lactose, or saccharine;and flavoring agents such as peppermint oil, oil of wintergreen, orartificial flavorings.

The autoinducer immunogenic conjugates or the autoinducer antibodies orbinding portions thereof of the present invention may also beadministered in injectable dosages by solution or suspension of thesematerials in a physiologically acceptable diluent with a pharmaceuticalcarrier. Such carriers include sterile liquids such as water and oils,with or without the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as polypropylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions. To maintain sterility and prevent action of microorganisms,antibacterial and antifungal agents, such as parabens, chlorobutanol,phenol, ascorbic acid, thimerosal, and the like may be added to thecarrier.

For use as aerosols, the immunogenic conjugates of the presentinvention, or antibodies or binding portions thereof according to thepresent invention, are alveolar lavage, lymph nodes, bone marrow, orother biopsied materials.

Toxicity and therapeutic efficacy of such molecules can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation).

The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from immuno-conjugation of multipleautoinducers with a carrier molecule.

In an embodiment, the autoinducer immunogenic conjugate vaccineformulation comprises an effective immunizing amount of the autoinducerimmunogenic conjugate, preferably in combination with animmunostimulant; and a pharmaceutically acceptable carrier. As used inthe present context, “immunostimulant” is intended to encompass anycompound or composition which has the ability to enhance the activity ofthe immune system, whether it be a specific potentiating effect incombination with a specific antigen, or simply an independent effectupon the activity of one or more elements of the immune response.Immunostimulant compounds include but are not limited to mineral gels,e.g. aluminum hydroxide; surface active substances such as lysolecithin,pluronic polyols; polyanions; peptides; oil emulsions; alum, and MDP.Methods of utilizing these materials are known in the art, and it iswell within the ability of the skilled artisan to determine an optimumamount of stimulant for a given autoinducer vaccine. More than oneimmunostimulant may be used in a given formulation. The immunogen mayalso be incorporated into liposomes, or conjugated to polysaccharidesand/or other polymers for use in a vaccine formulation.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration preferably foradministration to a human. Associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

5.4.2 Effective Dose

The compounds described herein can be administered to a patient attherapeutically effective doses to treat certain diseases caused by Gramnegative bacteria that cause infectious diseases. A therapeuticallyeffective dose refers to that amount of a compound sufficient to resultin a healthful benefit in the treated subject.

The precise amount of immunogenic conjugate or antibody whichspecifically binds a BAI to be employed in the formulation will dependon the route of administration and the nature of the patient (e.g., age,size, stage/level of disease), and should be decided according to thejudgment of the practitioner and each patient's circumstances accordingto standard clinical techniques. An effective immunizing amount is thatamount sufficient to treat or prevent an infectious disease caused by aGram negative bacteria that produces an autoinducer in a subject.Effective doses may also be extrapolated from dose-response curvesderived from animal model test systems and can vary from 0.001 mg/kg to100 mg/kg.

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from a cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Immunopotency of a composition can be determined by monitoring theimmune response of test animals following immunization with thecomposition by use of any immunoassay known in the art. Generation of ahumoral (antibody) response and/or cell-mediated immunity, may betaken-as an indication of an immune response. Test animals may includemice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., andeventually human subjects.

The immune response of the test subjects can be analyzed by variousapproaches such as: the reactivity of the resultant immune serum to theimmunogenic conjugate, as assayed by known techniques, e.g., enzymelinked immunosorbent assay (ELISA), immunoblots, immunoprecipitations,etc.; or, by protection of immunized hosts from infection by thepathogen and/or attenuation of symptoms due to infection by the pathogenin immunized hosts as determined by any method known in the art, forassaying the levels of an infectious disease agent, e.g., the bacteriallevels (for example, by culturing of a sample from the patient), etc.The levels of the infectious disease agent may also be determined bymeasuring the levels of the antigen against which the immunoglobulin wasdirected. A decrease in the levels of the infectious disease agent or anamelioration of the symptoms of the infectious disease indicates thatthe composition is effective.

The therapeutics of the invention can be tested in vitro, and then invivo, for the desired therapeutic or prophylactic activity, prior to usein humans. For example, in vitro assays that can be used to determinewhether administration of a specific therapeutic is indicated include invitro cell culture assays in which appropriate cells from a cell line orcells cultured from a patient having a particular disease or disorderare exposed to or otherwise administered a therapeutic, and the effectof the therapeutic on the cells is observed.

Alternatively, the therapeutic may be assayed by contacting thetherapeutic to cells (either cultured from a patient or from acultured-cell line) that are susceptible to infection by the infectiousdisease agent but that are not infected with the infectious diseaseagent, exposing the cells to the infectious disease agent, and thendetermining whether the infection rate of cells contacted with thetherapeutic was lower than the infection rate of cells not contactedwith the therapeutic. Infection of cells with an infectious diseaseagent may be assayed by any method known in the art.

In addition, the therapeutic can be assessed by measuring the level ofthe molecule against which the antibody is directed in the animal modelor human subject at suitable time intervals before, during, or aftertherapy. Any change or absence of change in the amount of the moleculecan be identified and correlated with the effect of the treatment on thesubject. The level of the molecule can be determined by any method knownin the art as described supra.

After vaccination of an animal to a Gram negative autoinducer using themethods and compositions of the present invention, any binding assayknown in the art can be used to assess the binding between the resultingantibody and the particular molecule. These assays may also be performedto select antibodies that exhibit a higher affinity or specificity forthe particular antigen.

5.5 Detection and Diagnostic Methods

Autoinducer antibodies or binding portions of the present invention areuseful for detecting in a sample the presence of an autoinducer of aGram negative bacteria. This detection method comprises the steps ofproviding an isolated antibody or binding portion thereof raised againstan autoinducer of a Gram negative bacteria, adding to the isolatedantibody or binding portion thereof a sample suspected of containing aquantity of the autoinducer, and detecting the presence of a complexcomprising the isolated antibody or binding portion thereof bound to theautoinducer.

The antibodies or binding portions thereof of the present invention arealso useful for detecting in a sample the presence of an autoinducerantagonist. This detection method comprises the steps of providing anisolated antibody or binding portion thereof raised against anautoinducer antagonist, adding to the isolated antibody or bindingportion thereof a sample suspected of containing a quantity of theautoinducer antagonist, and detecting the presence of a complexcomprising the isolated antibody or binding portion thereof bound to theautoinducer antagonist.

Immunoglobulins, particularly antibodies, (and functionally activefragments thereof) that bind a specific molecule that is a member of abinding pair may be used as diagnostics and prognostics, as describedherein. In various embodiments, the present invention provides themeasurement of a member of the binding pair, and the uses of suchmeasurements in clinical applications. The immunoglobulins in thepresent invention may be used, for example, in the detection of anantigen in a biological sample whereby patients may be tested foraberrant levels of the molecule to which the immunoglobulin binds,and/or for the presence of abnormal forms of such molecules. By“aberrant levels” is meant increased or decreased relative to thatpresent, or a standard level representing that present, in an analogoussample from a portion of the body or from a subject not having thedisease. The antibodies of this invention may also be included as areagent in a kit for use in a diagnostic or prognostic technique.

In an embodiment of the invention, an antibody of the invention thatimmunospecifically binds to an infectious disease agent may be used todiagnose, prognose or screen for an infectious disease associated withthe expression of the antigen of the infectious disease agent.

In a preferred aspect, the invention provides a method of diagnosing orscreening for the presence of an infectious disease agent, characterizedby the presence of an autoinducer antigen of a Gram negative bacteria ofsaid infectious disease agent, comprising measuring in a subject thelevel of immunospecific binding of an antibody to a sample derived fromthe subject, in which said antibody immunospecifically binds saidantigen in which an increase in the level of said immunospecificbinding, relative to the level of said immunospecific binding in ananalogous sample from a subject not having the infectious disease agent,indicates the presence of said infectious disease agent.

The measurement of a molecule that is bound by an antibody can bevaluable in detecting and/or staging diseases related to the molecule ina subject, in screening of such diseases in a population, indifferential diagnosis of the physiological condition of a subject, andin monitoring the effect of a therapeutic treatment on a subject.

Examples of suitable assays to detect the presence of autoinducers orantagonists thereof include but are not limited to ELISA,radioimmunoassay, gel-diffusion precipitation reaction assay,immunodiffusion assay, agglutination assay, fluorescent immunoassay,protein A immunoassay, or immunoelectrophoresis assay.

The following assays are designed to detect molecules to which theantibodies immunospecifically bind. The tissue or cell type to beanalyzed will generally include those which are known, or suspected, toexpress the particular molecule. The protein isolation methods employedherein may, for example, be such as those described in Harlow and Lane(Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The isolatedcells can be derived from cell culture or from a patient. The antibodies(or functionally active fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence, immunohistochemistry, or immunoelectron microscopy,for in situ detection of the molecule. In situ detection may beaccomplished by removing a histological specimen from a patient, such asparaffin embedded sections of affected tissues and applying thereto alabeled antibody of the present invention. The antibody (or functionallyactive fragment thereof) is preferably applied by overlaying the labeledantibody onto a biological sample. If the molecule to which the antibodybinds is present in the cytoplasm, it may be desirable to introduce theantibody inside the cell, for example, by making the cell membranepermeable. Through the use of such a procedure, it is possible todetermine not only the presence of the particular molecule, but also itsdistribution in the examined tissue. Using the present invention, thoseof ordinary skill will readily perceive that any of a wide variety ofhistological methods (such as staining procedures) can be modified inorder to achieve such in situ detection of Gram negative bacteriaautoinducers.

Immunoassays for the particular molecule will typically compriseincubating a sample, such as a biological fluid, a tissue extract,freshly harvested cells, or lysates of cultured cells, in the presenceof a detectably labeled antibody and detecting the bound antibody by anyof a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled antibody. The solidphase support may then be washed with the buffer a second time to removeunbound antibody. The amount of bound label on solid support may then bedetected by conventional means.

“Solid phase support or carrier” includes any support capable of bindingan antigen or an antibody. Well-known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given antibody may be determined according towell known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

One of the ways in which an antibody can be detectably labeled is bylinking the same to an enzyme and use in an enzyme immunoassay (EIA)(Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978,Diagnostic Horizons 2:1-7, Microbiological Associates QuarterlyPublication, Walkersville, Md.); Voller et al., 1978, J. Clin. Pathol.31:507-520; Butler, 1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.),1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa et al.,(eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo)). The enzyme whichis bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby calorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the syntheticantibodies or fragments, it is possible to detect the protein that theantibody was designed for through the use of a radioimmunoassay (RIA)(see, for example, Weintraub, 1986, Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety). The radioactive isotope can be detected by such means as theuse of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the syntheticantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems, in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

An additional aspect of the present invention relates to diagnostic kitsfor the detection or measurement of autoinducer immuno. Kits fordiagnostic use are provided, that comprise in one or more containers ananti-autoinducer antibody, and, optionally, a labeled binding partner tothe antibody. Alternatively, the anti-autoinducer antibody can belabeled (with a detectable marker, e.g., a chemiluminescent, enzymatic;fluorescent, or radioactive moiety). Accordingly, the present inventionprovides a diagnostic kit comprising, an anti-autoinducer antibody and acontrol immunoglobulin. In a specific embodiment, one of the foregoingcompounds of the container can be detectably labeled. A kit canoptionally further comprise in a container a predetermined amount of anautoinducer recognized by the said anti-autoinducer antibody of the kit,for use as a standard or control.

The Examples set forth below are for illustrative purposes only and arenot intended to limit, in any way, the scope of the present invention.

6. EXAMPLE Synthesis Preparation of Compound C

To a stirred solution of malonic acid mono tert-butylester A (419 mg,90% purity, 2.35 mmol) and L-homoserine lactone hydrochloride B (324 mg,2.35 mmol) in CH₂Cl₂ (15 mL) at room temperature was added sequentiallyEt₃N (660 μL, 4.94 mmol) andBenzotriazol-1-yloxytris(dimethylamino)phosphonium hexfluorophosphate(BOP) (1.14 g, 2.58 mmol). After it was stirred for 3 hours (h), themixture was concentrated, dissolved in EtOAc, washed with 1M HCl andsaturated NaHCO₃, dried over MgSO₄, filtered, concentrated and theresidual was chromatographed (SiO₂, Hexanes-EtOAc 1:1) to give C as agum (571 mg, 2.35 mmol, ca. 100%).

¹H NMR (300 MHZ, CDCl₃): δ 7.85 (br s, 1H), 4.61 (m, 1H), 4.50 (m, 1H),4.30 1(m, 1H), 3.32 (s, 2H), 2.81 (m, 1H), 2.23 (m, 1H), 1.50 (s, 9H).IR (cm⁻¹): 1780, 1716, 1683.

HRMS calculated for (C₁₁H₁₇NO₅+NH₄+) at 261.1450, found at 261.1453.

Preparation of Compound D

To a stirred solution of C (122 mg, 500 μmol) in CH₂Cl₂ (5 mL) at roomtemperature was added TFA (385 μL, 5 mmol), and the resulting mixturewas stirred for 4 h. The mixture was concentrated to give D as a gum (94mg, 500 μmol, ca. 100%).

¹H NMR (300 MHZ, CD₃CN): δ 9.70 (br s, 1H), 7.46 (br s, 1H), 4.60 (m,1H), 4.40 (m, 1H), 4.25 (m, 1H), 3.35 (s, 2H), 2.55 (m, 1H), 2.25 (m,1H). IR (cm⁻¹): 1773; 1731; 1656. HRMS calculated for (C₇H₉NO₅+NH₄ ⁺) at205.0824, found at 205.0829.

Preparation of Compound 5

To a stirred solution of L-homoserine lactone (Sigma, 1.0 mmol) in 10 mLof water at room temperature was added the lithium or sodium salt of thefatty acid (1.0 mmol) followed by1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Aldrich,1.0 mmol). After 24 h, the resulting milky suspension was extractedseveral times with ethyl acetate. The combined extracts were dried(MgSO₄), filtered and the solvent removed in vacuo. The crude amideswere then further purified by recrystallization from ethylacetate/hexane.

¹H NMR (CDCl₃, δ): 6.00 (bm, 1H), 4.46-4.69 (m, 2H), 4.25-4.34 (m, 1H),2.94-2.93 (m, 1H), 2.26 (t, J=7 Hz, 2H), 2.10-2.28 (m, 1H), 1.63-1.68(m, 2H), 1.27-1.31 (m, 10H), 09.89 (t, J=7 Hz, 3H). Anal. calcd forC₁₃H₂₃NO₃: C, 64.68; H, 9.62.

Found: C, 64.77; H, 9.72. [α]²⁵ _(D)+12° (c=2, CHCl₃), mp. 135-136° C.

Preparation of Compound 6

¹H NMR (acetone-d₆, +): 7.44 (bm, 1H), 4.55-4.65 (m, 1H), 4.55-4.65 (m,1H), 4.21-4.40 (m, 2H), 2.49-2.57 (m, 1H), 2.16-2.27 (m, 3H), 153-1.60(m, 2H), 1.26 (s, 16H), 0.85 (t, j=7 Hz, 3H). Anal. calcd for C₁₆H₂₉NO₃:C, 67.79; H, 10.33. Found: C, 67.87; H, 10.66.

Preparation of Compound 7

¹H NMR (CDCl₃, δ): 6.00 (bd, 1H), 4.45-4.60 (m, 2H), 4.25-4.34 (m, 1H),2.84-2.93 (m, 1H), 2.26 (t, J=7 Hz, 2H), 2.09-2.28 (m, 1H), 1.62-1.68(m, 2H), 1.26-1.30 (m, 22H), 0.89 (t, J=7 Hz, 3H). Anal. calcd forC₁₉H₃₅NO₃: C, 70.01; H, 10.86. Found: C, 69.48; H, 11.20. [α]²⁵ _(D)+12°(c=2, CHCl₃), mp. 138.139° C.

Preparation of Compound 8

To a stirred solution of Meldrums acid* (4.00 g; 27.8 mmol) and pyridine(5.62 mL; 69.4 mmol) in dry CH₂Cl₂ (12 mL) under N₂ at 0° C. was addedfreshly distilled decanoyl chloride (Aldrich, 5.76 mL; 27.8 mmol)dropwise. The reaction mixture gradually became deep red in color. After1 h the mixture was allowed to warm up to room temperature for 1.5 h. Atthis point, the mixture was poured over crushed ice containing 20 mL of2N HCl. The phases were separated and the aqueous layer furtherextracted with CH₂Cl₂. The combined organic extracts were dried (MgSO₄),filtered and concentrated in vacuo to give 3.882 g of crudebeta-ketoester 8.

* It is important to recrystallize the Meldrums acid from acetone beforecarrying out this experiment. The commercial material may be quiteimpure and adversely affect the purity of the final acylated product.

¹HNR (CDCl₃; δ):3.03 (m,2H), 1.75 (s, 6H), 1.62-1.70 (m, 2H), 1.25-1.35(m, 12H), 0.89 (t, j=7 Hz, 3H).

Preparation of Compound 10

The crude beta-ketoester from above was dissolved in 20 mL of drymethanol. The mixture was warmed to reflux under N₂. After 2.5 h, themixture was allowed to cool to room temperature and the methanol removedin vacuo. The oily residue was chromatographed (Eluent: 7% ethylacetate/hexane) to give 1.889 g (60%) of the beta-ketoester 9 as amixture of keto-enol tautomers. To a stirred solution of the β-ketoester9 (1.889 g; 8.3 mmol) in 20 mL of benzene at room temperature was addedethylene glycol (1.39 mL; 25 mmol) followed by 20 mg of p-TSA. Theresulting mixture was then warmed to reflux (Dean-Stark). After 24.5 h,the mixture was allowed to cool to room temperature and washed with 10%Na₂CO₃ solution. The organic layer was then dried (MgSO₄), filtered andconcentrated in vacuo. Flash chromatography on silica gel (eluent: 10%ethyl acetate/hexane) provided 1.926 g (85%) of the corresponding ketal10 as a clear oil.

¹H NMR (CDCl₃, δ): 3.99 (m, 4H), 3.70 (s, 3H), 2.68 (s, 2H), 1.77-1.82(m,2H), 1.35-1.41 (m, 2H), 1.27 (s, 14H), 0.89 (t, J=7 Hz, 3H). anal.calcd. for C₁₅H₂₈O₄: C, 66.13; H, 10.38. Found: C, 66.30; H, 10.18.

Preparation of Compound 11

To a stirred solution of the ketal of 9 in methanol at room temperaturewas added LiOH solution (3.65 mL of 1.0 mmol/mL stock solution). Themixture was then warmed to reflux for 10 min. and then allowed to coolto room temperature. The solvent was then removed in vacuo to give crudecarboxylate 10. The crude salt was then dissolved in 50 mL of water and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (844 mg;4.40 mmol) and L-homoserine lactone (556 mg; 4.04 mmol) were addedsequentially. The mixture was allowed to stir for 6 h at roomtemperature and then the resulting thick white suspension extractedseveral times with ethyl acetate. The combined extracts were dried(MgSO₄), filtered and concentrated in vacuo to give a crude off-whitesolid. Recrystallization from ethyl acetate/hexane afforded 752 mg (60%)of the amide 11.

¹H NMR (CDCl₃, δ): 7.01 (d, J=6 Hz, 1H), 4.56-4.61 (m, 1H), 4.47 (t, J=9Hz, 1H), 4.23-4.32 (m, 1H), 3.98-4.11 (m, 4H), 2.76-2.83 (m, 1H), 2.65(s, 2H), 205-2.223 (m, 1H), 1.66-1.75 (m, 4H), 1.26-1.42 (m, 2H), 1.26(s, 10H), 0.88 (t, 3=7 Hz, 3H). [α]²⁵ _(D)−16° (c=2, EtOH).

Preparation of Compound 13

To a stirred solution of the ketal 11 (330 mg; 0.97 mmol) in 2.0 mL ofmethanol at room temperature was added 0.1N HCl solution (2.0 mL). Theresulting cloudy mixture was warmed to reflux for 1 h and then allowedto cool to room temperature. The majority of the methanol was thenremoved in vacuo and the remaining solution extracted several times withethyl acetate. The combined extracts were dried (MgSO₄), filtered andconcentrated in vacuo. Flash chromatography on silica gel (eluent: 70%ethyl acetate/hexane) afforded 122 mg (43%) of PAI 13 as a waxy whitesolid mp. 88-90° C.

¹H NMR (CDCl₃, δ): 7.70 (bm, 1H), 4.58-4.64 (m, 1H), 4.49 (t, J=9 Hz,1H), 425-4.35 (m, 1H), 3.48 (s, 2H), 2.75-2.82 (m, 1H), 2.53 (t, =6 Hz,2H), 2.19-2.229 (m,1H), 1.57-1.65 (m, H), 1.27 (s, H), 0.89 (t, J=9 Hz,3H). Anal. calcd for C₁₆H₂₇O₄N: C, 64.60; H. 9.17. Found: C, 64.60; H,9.44. [α]²⁵ _(D)−19° (c=2, EtOH).

Preparation of Compound 12

To a stirred solution of the ester 10 (1.00 g; 3.67 mmol) in 5 mL ofmethanol at room temperature was added LiOH solution (3.55 mL of 1.0mmol/mL stock solution). The mixture was then warmed to reflux for 15min. and then allowed to cool to room temperature. The solvent was thenremoved in vacuo, the residue redissolved in 40 mL of water andL-homocysteine thiolactone hydrochloride (473 mg; 3.08 mmol) and1-(3-dimethylamonopropyl)-3-ethylcarbodiimide hydrochloride (590 mg;3.08 mmol) were added sequentially. The resulting mixture was allowed tostir for 19.5 h at room temperature and then extracted several timeswith ethyl acetate. The combined extracts were dried (MgSO₄), flit redand concentrated in vacuo. Flash chromatography on silica gel (eluent:85% ethyl acetate/hexane) provided 585 mg. (64%) of thethiolactone-amide 12 as a white solid.

¹H NMR (CDCl₃, δ): 6.92 (bd, 1H), 4.57-4.62 (m, 1H), 3.98-4.12 (m, 4H),3.24-340 (m, 2H), 2.87-2.93 (m, 1H), 2.65 (s, 2H), 1.92-2.00 (m, 1H),1.66-1.75 (m, 2H), 1.32-1.40 (m, 2H), 1.27 (s, 12H), 0.89 (t, J=7 Hz,3H).

Preparation of Compound 14

To a stirred solution of the ketal 12 (487 mg; 1.36 mmol) in 12.0 mL ofmethanol at room temperature was added 0.1N HCl solution (7.0 mL). Theresulting cloudy mixture was warmed to reflux for 30 min. and thenallowed to cool to room temperature. The majority of the methanol wasthen removed in vacuo and the remaining solution extracted several timeswith ethyl acetate. The combined extracts were dried (MgSO₄), filteredand concentrated in vacuo. Flash chromatography on silica gel (eluent:85% ethyl acetate/hexane) provided 323 mg (76%) of the keto-amide 14 asa white solid, mp. 86-88° C.

¹H NMR (CDCl₃, δ): 7.51 (bd, 1H), 4.60 (m, 1H), 3.47 (s, 2H), 3.24-3.44(m,2H), 2.82-2.90 (m, 1H), 2.54 (t, J=7 Hz, 2H), 1.98-2.09 (m, 1H),1.57-1.61 (m,2H), 127 (s, 12H), 0.89 (t, J=7 Hz, 3H). Anal. calcd forC₁₆H₂₇NO₃S: C, 61.29; H, 8.70. Found: C, 61.21; H, 8.75. [α]²⁵ _(D)−19°(c=2, EtOH).

Preparation of Compound 15

To a stirred solution of PAI 13 (70 mg; 0.24 mmol) in 3.0 mL of drymethanol under N₂ at −15° C. was added sodium borohydride (10 mg; 0.26mmol). After 1 h, another 10 mg of sodium borohydride was added andstirring allowed to continue for another hour. At this point, themixture was quenched with 1 mL of acetone and allowed to warm to roomtemperature. The solvent was removed in vacuo, the residue redissolvedin CH₂Cl₂ and washed with water. The organic layer was then dried(MgSO₄), filtered and concentrated in vacuo. The crude solid wasrecrystallized from ethyl acetate/hexane to give 39 mg. (55%) of thehydroxy-amide 15 as a mixture of 2 diastereomers.

¹H NMR (CDCl₃, δ): 6.48-6.60 (bm, 1H), 4.46-4.64 (M, 2H), 4.27-4.38 (m,1H), 4.00-4.06 (m, 1H), 2.81-2.90 (m, 1H), 2.08-2.52 (m, 5H), 1.39-1.58(m, 2H), 128 (s, 12H).

Preparation of Compound 17

To a stirred solution of the oxazolidinone 16 (Fluka, 388 mg; 3.0 mmol)in 10 mL of dry THF under N₂ at −78° C. was added n-BuLi solution (1.38mL of 2.4 mmol/mL solution) dropwise. At the end of the addition, themixture was warmed to 0° C. for 10 min., quenched with decanoyl chloride(0.81 mL; 3.9 mmol) and stirred for an additional 30 min. at thistemperature. At this point, the mixture was quenched with 2.0 mL ofsaturated NaHCO₃ solution and the solvent removed in vacuo. The residuewas taken up into CH₂Cl₂ and washed with 20% K₂CO₃ solution. Theorganic-layer was then dried (MgSO₄), filtered and the solvent removedin vacuo. Flash chromatography on silica gel (eluent: 35% ethylacetate/hexane) provided 740 mg. (87%) of oily oxazolidinone 17.

¹H NMR (CDCl₃, δ): 4.43-4.48 (m, 1H), 4.20-4.31 (m, 2H), 2.83-3.03 (m,2H), 2.36-2.42 (m, 1H), 1.62-1.69 (m, 2H), 1.28-1.33 (m, 12H), 0.88-0.94(m, 9H), Anal. calcd for C₁₆H₂₉O₃N: C, 67.79; H, 10.33. Found: C, 67.87;H, 10.59. [α]²⁵ _(D+)69° (c=2, EtOH).

Preparation of Compound 21

To a stirred solution of trans-4-decanal (2.00 g; 13.0 mmol) andsulfamic acid (3.78 g; 38.9 mmol) in 80 mL of 3:1 THF:H₂O at 0° C. wasadded NaClO₂ solution (1.41 g; 15.6 mmol dissolved in 10 mL of water)dropwise. At the end of the addition, the mixture was allowed to warm toroom temperature for 10 min. At this point, most of the THF was thenremoved in vacuo. The resulting mixture was extracted several times withCH₂Cl₂. The combined extracts were dried (MgSO₄), filtered andconcentrated in vacuo. The residue was diluted with 10 mL of hexane,filtered and finally concentrated in vacuo to give 2.342 g of crude oilyacid 18. To a stirred solution of the crude acid (2.342 g) in 50 mL ofdry CH₂Cl₂ under N₂ at room temperature was added 2 drops of DMFfollowed by oxalyl chloride (1.56 mL; 17.9 mmol) dropwise. After 30min., gas evolution had ceased and the solvent was removed in vacuo toprovide the crude acid chloride 19. Crude 19 was then redissolved in 30mL of CH₂Cl₂ and Meldrums acid (1.99 g; 13.8 mmol) was added. Themixture was cooled to 0° C. and pyridine (2.79 mL; 34.5 mmol) wasdropwise. After the addition was complete, the mixture was allowed towarm to room temperature for 1.5 h then diluted with CH₂Cl₂ and washedwith 10% KHSO₄ solution. The organic layer was then dried (MgSO₄),filtered and concentrated in vacuo. The residue was then dissolved in 30mL of methanol and heated to reflux for 1 h, cooled and the solventremoved in vacuo. The oily residue was chromatographed on silica gel(eluent: 8% ethyl acetate/hexane) to give 558 mg (18%) of thebeta-ketoester 21 as a clear oil.

Keto tautomer: ¹H NMR (CDCl₃, δ): 5.37-4.58 (m, 2H), 3.76 (s, 3H), 3.47(s, 2H), 2.62 (t, J=7 Hz, 2H), 2.27-2.35 (m, 3H), 1.94-2.01 (m, 2H),1.23-1.38 (m, 6H), 0.90 (s, 3H).

Preparation of Compound 22

To a stirred solution of the beta-ketoester in benzene at roomtemperature was added ethylene glycol (0.64 mL; 11.4 mmol) followed by atrace of p-TSA. The mixture was then warmed to reflux Dean-Stark). After4 h, another 0.64 mL of ethylene glycol was added. After a total of 23h, the mixture was allowed to cool to room temperature and washed with10% Na₂CO₃ solution. The organic layer was then dried (MgSO₄), filteredand concentrated in vacuo. Flash chromatography on silica gel (eluent:8% ethyl acetate/hexane provided 513 mg (50%) of the ketal intermediate22 together with 152 mg (18%) of recovered beta-ketoester 21.

¹H NMR (CDCl₃, δ): 5.39-5.49 (m, 2H), 3.99-4.01 (4H), 3.71 (s, 3H), 2.69(s, 2H), 1.86-2.15 (m, 7H), 1.29-1.37 (m, 6H), 0.90 (t, J=7 Hz, 3H).

Preparation of Compound 24

To a stirred solution of the ketal (450 mg; 1.66 mmol) in 4.0 mL ofmethanol at room temperature was added LiOH solution (1.66 mL of 1.0 Mstock solution). The mixture was then warmed to reflux for 15 min. andthen allowed to cool. The solvent was removed in vacuo and the crudecarboxylate redissolved in 20 mL of water. To this solution was addedL-homoserine lactone hydrochloride (251 mg; 1.83 mmol) followed by1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (350 mg;1.83 mmol) and the mixture allowed to stir for 6 h at room temperature.At this point, the mixture was extracted several times with ethylacetate. The combined extracts were dried (MgSO₄), filtered andconcentrated in vacuo to give 531 mg of crude amide 24.

¹H NMR (CDCl₃, δ): 7.00 (d, J=6 Hz, 1H), 5.38-5.44 (m, 2H), 4.55-4.65(m, 1H), 4.45-4.51 (m, 1H), 4.28-4.31 (m, 1H), 3.99-4.12 (m, 4H),2.79-2.83 (m, 1H), 2.67 (s, 2H), 1.74-2.19 (m, 7H), 1.29-1.36 (m, 6H),0.89 (t, J=7 Hz, 3H).

Preparation of Compound 25

The crude amide 24 was dissolved in 4.0 mL of methanol at roomtemperature and 3.0 mL of 0.1 N HCl solution was added. The resultingmixture was warmed to reflux for 1 h, cooled to room temperature and themajority of the methanol removed in vacuo. The remaining aqueous phasewas extracted several times with ethyl acetate. The combined extractswere dried (MgSO₄), filtered and concentrated in vacuo. Flashchromatography on silica gel (eluent: 75% ethyl acetate/hexane) gave 175mg (36%) of the unsaturated keto-amide 25, mp. 92-93° C.

¹H NMR (CDCl₃, δ): 7.70 (d, J=4.3 Hz, 1H), 5.32-5.50 (m, 2H), 4.56-4.65(m, 1H), 4.46-4.52 (m, 1H), 4.25-4.33 (m, 1H), 3.48 (s, 2H), 2.72-2.81(m, 1H), 2.58-2.63 (m, 2H), 2.17-2.32 (m, 3H), 1.93-1.98 (m, 2H),1.25-1.38 (m, 6H), 0.89 (t, J=7 Hz, 3H). [α]²⁵ _(D)−23° (c=2, EtOH).

Preparation of Compound 27

Prepared by coupling of the aminolactam 26 (129 mg; 0.94 mmol) and thelithium salt derived from the ester 10 (278 mg; 1.02 mmol) in water (2.0mL) in the presence of carbodiimide 4 (180 mg; 0.94 mmol) according tothe procedure used for the preparation of compound 11. The crude product(293 mg) was then dissolved in 7.0 mL of methanol and 3.0 mL of 0.1 NHCl was added and the mixture heated to reflux for 1 h and then allowedto cool to room temperature and then processed as for compound 14. Thecrude solid product was recrystallized from ethyl acetate/hexane to give150 mg (54%) of amide 27, mp. 158-160° C.

¹H NMR (CDCl₃, δ): 7.60 (bm, 1H), 6.45 (bm, 1H), 4.45 (m, 1H), 3.47 (s,2H) 3.338-3.42 (m, 2H), 2.69-2.77 (m, 1H), 2.55 (t, J=7 Hz, 2H),1.96-2.04 (m, 1H), 1.58-1.61 (m, 2H), 1.27 (s, 12H), 0.89 (t, J=7 Hz,3H).

Preparation of Compound 29

A solution of maleic anhydride (12.2 mg, 0.125 mmol) in dichloromethane(0.2 mL) was added to a solution of DL homoserine lactone (12.6 mg,0.125 mmol) in dichloromethane (0.1 mL). As the mixture was stirred, awhite precipitate formed rapidly, became oily over 10 min, then became agranular solid over the next 10 min. Stirring was continued for a totalof 75 min, and the mixture was then diluted with di-isopropyl ether (2mL) and the solid was removed by filtration to give N-maleoylhomoserinelactone (20 mg, 81%).

¹H NMR (DMSO) 13.3 (br s, 1H), 9.12 (br d, J=8.8 Hz, 1H), 6.30, 6.24 ( 2d, J=12.2 Hz, 1H each), 4.64 (ddd, J=10.5, 9.1, 7.9 Hz, 1H), 4.36 (ddd,J=8.8, 8.8, 1.9 Hz, 1H), 4.23 (ddd, I=10.5, 8.7, 6.5 Hz, 1H), 2.43(dddd, J=12.2, 9.1, 6.4, 1.9 Hz, 1H), 2.19 (dddd, J=12.2, 8.8, 8.7, 7.9Hz, 1H). ¹³C NMR (DMSO) 174.8(s), 166.5(s), 164.8(s), 131.7(d),130.2(d), 65.5(t), 48.2(d), 21.8(t).

7. EXAMPLE Production of BAI Antibodies

Production of anti-autoinducer antibodies and experimental in vivotherapeutic evaluation of said anti-autoinducer antibodies is describedin the sections that follow. Specifically, antibodies were made toimmunogenic conjugates containing PAI-1 covalently bound to bovine serumalbumin, however, one of ordinary skill in the art will easily recognizethat other autoinducer molecules and additional conjugates may be usedin a similar procedure to produce various combinations of immunogenicconjugates and antibodies that recognize said immunogenic conjugates.One of ordinary skill in the art will also recognize that additionalanimal models can be used to assay the therapeutic and diagnosticeffectiveness of the anti-autoinducer antibodies.

7.1 PAI'S Role in Virulence

The role of PAI-1 and PAI-2 in colonization of Pseudomonas aeruginosa inneonatal mice was examined. BALB/cBy, mice 7-10 days old, wereinoculated intranasally with approximately 10⁸ CFU of either wild typeor deletion mutants of P. aeruginosa. The lasI gene product is asynthase required for PAI-1 production and the rhII gene product isrequired for production of PAI-2 (Passador et al., 1996, J. Bact.178:5995-6000). The deletion strains were: PAO1, wild type; PAO-JP2,bearing a double deletion of lasI/rhII; PAO-JP1, bearing a lasIdeletion; and PAO-JP2/pJPP42, bearing a double deletion lasI/rhII butcarrying a complementing plasmid expressing both lasI and rhII.Bacterial suspensions of these strains were given in 2 μl aliquotsdirectly into the mouse nares until a total of 10 μl had beenadministered. Twenty-four hours after challenge, mice were sacrificed,and the right lung cultured for bacterial load and the left lung fixedin 10% buffered formalin for histological analysis. Pneumonia wasdefined as ≧100 CFU of Pseudomonas aeruginosa in the lung. Bacteremiawas defined as any bacteria cultured from the spleen.

The results indicate that mice inoculated with strain PAO-JP1, whichdoes not produce PAI-1, exhibited a significant decrease in the rates ofpneumonia, bacteremia, and mortality in comparison to PAO1 (FIG. 1).Moreover, mice inoculated with strain PAO-JP2, which produces neitherPAI-1 nor PAI-2, exhibited an additional decrease in the rates ofpneumonia, bacteremia, and mortality over the already reduced infectionrate of PAO1. In sharp contrast, mice inoculated with Pseudomonasaeruginosa JP2/pJPP42, which produces PAI-1 and PA-2 by virtue of agenetically complementing plasmid, exhibited pneumonia, bacteremia, andmortality rates comparable to wild type, PAO1 (FIG. 1).

7.2 Anti-PAI Polyclonal Antibodies

The Pseudomonas aeruginosa autoinducer PAI-1 (structure on page. 16) andCompound D (structure on page 9) were separately conjugated to bovineserum albumin (BSA). These conjugates were used to immunize differentBALB/c mice. Initial injections of 50 μg of conjugate were administeredsubcutaneously in Freund's complete adjuvant and two weeks later wereboosted with a second subcutaneous injection in Freund's incompleteadjuvant. After two additional weeks, mice were tail bled and serum wasisolated. A sandwich enzyme-linked immunosorbent assay (ELISA) utilizinga PAI-1 ovalbumin (OVA) conjugate was used to test for the production ofspecific serum antibodies that recognize autoinducers. Serum sampleswith an optical density (OD) that was five to ten fold higher than serumfrom mice immunized with BSA only were considered positive.

7.3 Neutralization of PAI with Antibodies

In Vitro neutralization of PAI-1 with anti-PAI-1 polyclonal antibodies.Immune serum, collected in Section 7.2, were tested for neutralizationof PAI-1 in an in vitro bioassay. The E. coli MG4 strain, containing thelysogen λI₁4 (a lasI/lasZ transcriptional fusion) and pPCS1 (a plasmidexpressing lasR), was used as a positive control to detect the presenceof PAI-1. Normally, when PAI-1 is added to cultures it can bind LasR,the PAI-1 specific transcriptional activator protein, and form a complexthat is able to induce transcription of the lasI/lasZ fusion protein.The production of β-galactosidase in this system is a quantitative anddirect measure of the activation induced by PAI-1. The expressionconstruct, λI₁4-MG4 (pPCS1), has been shown to have half-maximalexpression at PAI-1 concentrations of 100 pM and can be activated atPAI-1 concentrations as low as 10 pM (Seed et al., 1995, J. Bacteriol.177:654-59, which is hereby incorporated by reference).

A test sample containing 100 pM of PAI-1 was preincubated for one hourat 37° C. with a 1:10 dilution of serum from mice immunized with a PAI-1conjugate or a Compound D conjugate (both contain anti-PAI-1 polyclonalantibodies). Control samples containing 100 pM PAI-1 were incubated at37° C. for one hour with a 1:10 dilution of preimmune serum or an equalvolume of PBS. Following preincubation, the samples were tested in an E.coli bioassay using λI₁4-MG4 (pPCS1). When the bacteria in each samplereached an OD600 of 0.8-1.0 the samples were assayed for the productionof β-galactosidase which was expressed as Miller Units of activity. Thetest samples preincubated with serum from immune mice displayed a 70%reduction in β-galactosidase production as compared to control samplepreincubated with nonimmune serum (FIG. 2). These results indicate thatPAI-1 conjugates or related conjugates (Compound D) can induce theproduction of polyclonal antibodies that can react with PAI-1 andinhibit its interaction with LasR.

Mice immunized with the PAI-1 conjugate were used to produce monoclonalantibodies. These antibodies were screened using an ELISA utilizing aPAI-1/OVA conjugate. Positive clones were tested in a Pseudomonasaeruginosa bioassay. PAO-JP2 (bearing the lasI/rhII double deletion)produces no PAI-1 but retains the ability to produce LasR. When PAI-1 isadded exogenously, it can bind to LasR and induce the transcription oflasI. Test samples containing 40 nM PAI-1 (the concentration thatstimulates half-maximal activity in this assay) were preincubated at 37°C. with anti-PAI-1 monoclonal antibody (618.4). Control samplescontaining 40 nm PAI-1 were preincubated at 37° C. with an equal volumeof PBS. Following preincubation, the samples were tested in aPseudomonas aeruginosa bioassay using PAO-JP2 containing a plasmid witha lasI/LacZ fusion. When the bacteria reached on OD₆₀₀ of 0.8-1.0, thesamples were assayed for the production of β-galactosidase, which wasexpressed as Miller Units of activity. The test sample preincubated withanti-PAI-1 monoclonal antibody 618.4 displayed an 80% reduction in theproduction of β-galactosidase as compared to the control sample (FIG.3). These results indicate that in Pseudomonas aeruginosa antibodiesspecific for PAI-1 can inhibit PAI-1 activation of LasR andtranscription of other genes that are regulated by LasR/PAI-1.

Although the invention has been described in detail for the purposes ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method for detecting an autoinducer of a Gram negative bacterium ina sample comprising: adding to the sample an antibody or a functionallyactive fragment thereof which binds immuno specifically to theautoinducer of the Gram negative bacterium having Formula (I):

wherein X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straightor branched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl C₁-C₁₈ straight or branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkyl,C₁-C₆ straight or branched alkenyl or C₁-C₆ straight or branchedalkynyl, or CO₂H; and Q is CH or N; and n is 0-3 with the proviso thatwhen n is 0, X is N—(C₁-C₆ alkyl) or N-phenyl; and detecting whetherimmonospecific binding occurs between the antibody, or the functionallyactive fragment thereof, and the autoinducer of the Gram negativebacterium, wherein said immunospecific binding indicates presence of theautoinducer in the sample.
 2. The method according to claim 1 whereinthe Gram negative bacterium is selected from the group consisting ofAerononas hydrophila, Agrobacterium tumefaciens, Burkholderia cepacia,Chromobacterium violaceum, Enterobacter agglomerans, Erwinia stewarti,Erwinia carotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, and Vibrio harveyi.
 3. The methodaccording to claim 1 wherein the antibody or the functionally activefragment thereof comprises a label.
 4. The method according to claim 3wherein the label is an enzyme, a radioactive label, a fluorescentlabel, a chemiluminescent compound, or a bioluminescent compound.
 5. Themethod according to claim 1 wherein said detecting is carried out byELISA, radioimmunoassay, gel-diffusion precipitation reaction assay,immunodiffusion assay, agglutination assay, fluorescent immunoassay,protein A immunoassay, or immunoelectrophoresis assay.
 6. The methodaccording to claim 1 wherein the sample is a histological sampleobtained from a patient.
 7. The method according to claim 6 wherein saiddetecting is carried out in situ.
 8. The method according to claim 1wherein the sample is selected from the group consisting of biologicalfluids, tissue extracts, freshly harvested cells, and lysates ofcultured cells.